The biotechnical revolution, including development of modern biopharmaceuticals and mapping of the human genome, has been made possible due to development of separation methods such as chromatography and electrophoresis. Such methods can be used in small scale as well as in large scale, and are known as flexible methods, being useful for a variety of substances including biological substances. However, they are demanding both technically and in terms of equipment. In addition, scaling of some processes such as preparative electrophoresis results in a need for more complicated equipment due to nonlinear scaling of heating and cooling requirements. Such complications also hinder modelling of such methods and their optimisation via (small volume, microtiter) high throughput screening methods.
Partitioning between the phases in aqueous polymer phase systems is an alternative method, which has been studied since the 1950's but whose commercial application has been severely restrained by lack of economically feasible (inexpensive) phase systems offering good capacity (target solubility). Together with separation methods such as flocculation, crystallization and size exclusion; partitioning is considered a classic separation technique. It is related to differentially distributing a target and other substances between two phases. The term “partitioning” can refer to (a) liquid-solid partition such as in classic capture chromatography, (b) partitioning between two or more liquid phases (biphasic and multiphase system, respectively), (c) partitioning between a mobile liquid phase and another liquid phase immobilized at the surface of a solid phase support, and (d) partitioning of particles between a liquid phase and the phase interface between two phases. For the purposes of this patent application, partition and partitioning refer to situations such as b, c or d i.e. partitioning between liquid phases. In this definition target capacity is not as much a function of (solid) phase surface area as much as liquid phase volumes. As a result capacities can be very high (see below). Partition is typically expressed as a coefficient (K) related to the concentration in one phase versus another and for solutes K generally follows the Brønsted equation. Thus K is expected to vary exponentially with various types of interactions such as electrostatic and/or hydrophobic interactions, and also to be sensitive to solute size i.e. the area of interaction with liquid phases. In the case of interfacial partition, where particles may be held at a liquid-liquid phase interface by interfacial tension, K is expected to vary exponentially with interfacial tension, as well as phase compositional factors.
Classic liquid-liquid two-phase systems are organic and aqueous two phase systems which normally have significant polarity differences between the phases, as well as significant interfacial tension. Such systems are not very useful for biologicals, such as proteins or cells, as they tend to be denatured by significantly apolar solutions and shear damage related to mixing of phase systems with significant interfacial tension. More useful for biologicals are low tension, aqueous polymer two phase-systems. It is well recognized that the latter may contain some added organic solvents, e.g. ethanol, or other organic additives added to enhance target solubility, reduce liquid phase polarity, reduce foaming, act as bactericidal agents, etc.
Polymer two-phase systems can be formed by mixing certain hydrophilic and typically neutral polymers in aqueous solution. These include dextran (polyglucose) and poly(ethylene glycol) (PEG); as well as polysucrose (such as Ficoll™) and PEG; or linear polyacrylamide and PEG. Typical concentrations of each polymer are 5 to 10% w/w. At such concentrations, entropic and other forces tend to drive the formation of two phases both of which are typically greater than 90% (w/w) water but show subtle differences in polarity, hydrogen bond character, freezing point, etc. The phases are typically enriched in one polymer and have low interfacial tension. Phase density differences drive the phases to separate by gravity or centrifugation. In the biotechnical field, one advantage of the PEG and Dextran type of two-phase system is that target proteins may partition in favour of the PEG-enriched, less dense, upper phase while cell debris and some contaminants may partition (or sediment) to the interface or complementary lower phase.
Independent of the challenging of adding and then removing two polymers from a bioprocess stream the major drawback to dextran and PEG and similar two-polymer phase systems is the cost of the polymers. This is especially true for the dextran, a natural bioproduct which must itself be purified for use in bioprocess phase systems. In an effort to reduce such costs scientists have investigated two paths. The first is to replace dextran with starch or other less expensive polymers. However such polymers often are less pure, less controlled in MW, form more viscous phases and come with their own unique challenges (Josefine Persson, Dana C. Andersen, Philip M. Lester, Biotechnology and Bioengineering, vol. 90, (2005) 442-451). The other approach has been to work with two phase systems formed by combining relatively high concentrations of PEG (10% w/w) and salts such as potassium sulphate (3% w/w). In regard to protein partition in such systems see Andrews, Nielsen, Asenjo, 1996. and Azevedo et al., 2007 (discussed in more detail below) while for recent review related to plasmid partition see F. Rahimpour, F. Feyzi, S. Maghsoudi, R. Hatti-Kaul, Biotechnology and Bioengineering, 95, 627-637, 2006.) Unfortunately the increased PEG and salt concentrations create challenges which negatively impact process costs. These include viscous phases, salt reagent costs, salt disposal and equipment corrosion challenges, as well as target solubility issues which relate to capacity. For example antibody capacity in these systems is often 1 g/L which means (clarified) fermentation broth containing expressed antibody at 10 g/L would have to be diluted ten fold prior to partitioning. It also means that if the phase systems cost five dollars a liter to formulate then they add at least five dollars a gram to the cost of goods. Such dilution and related increases in process volumes, process times and costs are prohibitive.
Some hydrophilic polymers exhibit inverse thermal solubility such that as temperature is raised above a certain cloud temperature (Tc) which is related to a polymer's lower critical solubility temperature (LCST), they self associate and start to form a unique polymer rich phase. Common literature offers several examples of such polymers including copolymer or block copolymers formed with mixtures of ethylene oxide (EO) and propylene oxide (PO) monomeric groups, so called EOPO polymers, polysaccharides modified with EO, PO or similar groups (e.g. ethylhydroxyethylcellulose or EHEC), or polymers formed using N-isopropylacrylamide (NIPAAM). Whereas the Tc for PEG (polymerised EO) in dilute buffered solution is around 100° C., and thus unsuitable for most biotechnical applications, the Tc for EOPO and NIPAAM polymers is often in more biotechnically useful range of 20 to 40° C., depending on solution salt composition and other factors. In addition to thermoresponsive polymers some hydrophilic polymers exhibit pH dependent self association (e.g. WO 2004/082801 A1). WO 2004/020629 (Tjerneld) relates to the use of the EOPO polymers' reverse thermal solubility to further facilitate the separation of plasmids already partitioned in a two polymer phase system. At room temperature the two-polymer, two phase system formed with EOPO and dextran polymers forms in same manner as PEG and dextran system. The less dense EOPO-enriched upper phase is isolated from the EOPO and dextran polymer aqueous two-phase system. The temperature of the EOPO-enriched phase is then raised to approximately 37° C. (i.e. above Tc) so that the upper phase undergoes further phase separation into a water-enriched phase and a self-associated EOPO polymer-enriched phase. Advantageously, the water-enriched phase should contain the desired target. In general, these kinds of EOPO and dextran systems offer advantages in terms of phase polymer component recycling and design of efficient two-stage partition separation process. However, a drawback is again the cost involved in system formulation using the biologically derived and costly dextran polymer. Less expensive polymers such as starch polymers may replace dextran in such systems (Persson et al, 2005) but there are still challenges associated with having to add then remove two polymers from the process stream.
In the above literature examples, as in the general literature, phase systems are used to purify targets from clarified feed formed by subjecting phase fermentation broth containing intact or lysed cells and cell debris to centrifugation.
In the biotechnical field, aqueous polymer two phase systems, formed with two polymers or with one polymer in presence of significant added salt are of general interest. This is because they are easily utilised in small as well as larger scale separations, without loss of efficiencies or dramatic changes in costs when scaling up to the larger volumes. Also, any standard separation approach, such as charge-based, hydrophobicity-based, affinity-based, or size-based separation, can be performed within a polymer two phase system. In general many undesired components, such as cell debris, endotoxins, nucleic acids, virus, will tend to appreciably partition to the lower (dextran-rich or salt rich, respectively) phase in a PEG and dextran, or a PEG and salt two phase system. Thus, if a system can be found which provides for good target partition into the upper (PEG-rich) phase an effective primary separation and target concentration can be obtained. However four major hurdles will still remain in terms of capacity (i.e. solubility), phase component cost, phase component removal, and effect of phase components on other (downstream) unit operations and equipment. The latter particularly inhibits easy incorporation of some phase systems, as upstream unit operations, in existing standard processes.
In efforts to overcome drawbacks related to interfacing in standard chromatographic and/or filtration processing, and to overcome the limitations of a single theoretical partition step per unit operation, liquid-liquid partitioning two phase systems such as PEG-dextran or PEG-salt have been adapted to chromatographic uses by immobilising one phase on a chromatographic or other solid support capable of preferentially wetting that phase. The complementary phase is then pumped through the column offering repeated opportunities for equilibration between the mobile and stationary phase. This was commercially exploited by W. Müller et al. at E. Merck, Darmstadt in the 1980's (U.S. Pat. No. 4,756,834).
Various combinations of the above approaches and other phase forming polymers are possible. U.S. Pat. No. 5,093,254 (Giuliano et al) relates to an aqueous two-phase protein partitioning system which employs polyvinylpyrrolidone (PVP) as the upper phase and maltodextrin as the lower phase and provides a low-cost system for protein partitioning. The system can also be employed with certain derivatives of chlorotriazine dyes, which bind in a noncovalent manner to the PVP and serve as a ligand for the proteins to be separated. It is stated that an advantage of this system is its cost-efficiency, as the dyes can easily be bound to the polymeric phase, without having to carry out the chromatographic and solvent extractions necessary to form the covalent bond in the PEG and hydroxypropyl starch system of the prior art.
Many modern biopharmaceuticals are based on monoclonal antibodies (typically IgG forms) or related antibody fragments (Fabs) or derivatives of antibodies. Use of phase systems for purification of antibodies has been studied for over thirty years, if one includes studies of plasma protein partitioning in dextran and PEG and related two polymer biphasic systems. Studies directed towards feasibility of large scale processing of antibodies by partitioning, using more cost effective PEG-salt and other systems have been in the literature for over a decade.
B. A. Andrews, S. Nielsen and J. A. Asenjo (Partitioning and purification of monoclonal antibodies in aqueous two-phase systems, Bioseparation 6, (1996) 306-313) investigated systems and used factorial design to find some they consider optimal for antibody partitioning such as 7% w/w PEG 1450, 14% NaPhosphate and 12% NaCl. Such systems gave antibody partition K (ratio of protein concentration in upper phase over lower phase) values of 100. They used serum albumin, transferin and some other proteins to represent process feed stream contaminants and demonstrated differential partition to that shown for antibodies. In addition they attempted small scale processing of a monoclonal antibody sample from hybridoma cell culture. As with Persson et al. 2005 and Azevedo et al., 2007, they worked with centrifuge clarified (cell free) sample solutions. In the experiments with hybridoma produced antibody sample they noted that K values obtained with pure protein samples appeared compromised by sample solution complexity. However they were able to achieve good partition of antibody into one phase, and show ability to enhance purity using multiple extractions, including those where the target molecule is partitioned into complementary phase using a system with lower NaCl.
Andrews et al. also noted what remains the main drawback to PEG salt system protein partitioning in general, and antibody partitioning in particular, which (due to the high salt concentrations) is low protein solubility (often 1 g/L). If one considers that antibodies and other recombinant proteins may be expressed at levels of 10 g/L or higher use of such systems early in separation process would entail a 10-fold increase in process volume with a several fold increase in processing scales, costs and times. In addition to these costs would be those related to salt components including salt removal and possible corrosion of pumps and other metal equipment. A decade later Azevedo et al. (Ana M. Azevedo, Paula A. J. Rosa, I. Filipa Ferreira, M. Raquel Aires-Barros, Optimisation of aqueous two-phase extraction of human antibodies, Journal of Biotechnology 132 (2007) 209-217) extended efforts to find PEG and salt systems suitable for industrial scale process of antibodies. Their optimisation methods found systems similar to those of Andrews et al. (i.e. 12% PEG 6000, 10% NaPhosphate pH 6, 15% NaCl) which when used to partially purify Mab from a concentrated (and clarified) Chinese Hampster Ovary (CHO) cell culture supernatant with total yield of 88% and from hybridoma culture supernatant with a total yield of 90%. However their target protein concentrations were still approximately 1 g/L.
More recently Aires-Barros et al (I. Filipa Ferreira, Ana M. Azevedo, Paula A. J. Rosa, M. Raquel Aires-Barros, Purification of human immunoglobulin G by thermoseparating aqueous two-phase systems, Journal of Chromatography A, 1195 (2008) 94-100) have investigated two polymer thermoseparating phase systems for antibody partitioning in systems containing UCON® EOPO 50/50 copolymers of MW 2000 to 5100 (Dow Chemical). They studied partitioning of IgG from clarified CHO culture supernatant (Ab at 0.1 g/L) between the phases in 8% w/w UCON and 5% dextran T500 systems and to enhance antibody partition into the upper (EOPO polymer-rich) phase they added 20% w/w triethylene glycol-diglutaric acid (TEG-COOH) and 10 mM NaPhosphate pH 8. Clarified supernatant could be added to systems at 50% (to achieve above final polymer and TEG-COOH concentrations). In some experiments polyclonal IgG (Gammanorm™, Octapharma AG) was added to increase target protein to approximately 1 g/L. A two step (two polymer two phase partition followed by thermoseparation of the upper phase into polymer-rich and water rich phases, see above) partition process yielded 85% of antibody (which is relatively low for a commercially attractive process) at 88% purity (which may have been aided by adding in GammaNorm). Tc occurred at approximately 50° C. which required applied heating of the phase system in the second step extraction. While these systems offer lower salt concentration they also require significant TEG-COOH as in its absence recovery yield of IgG in the top UCON-rich phase (of UCON and Dextran phase system) was lower than 50% (i.e. K<1).
In general thermoseparating phases have normally been used together with dextran (see Aires-Barros et al, above) or similar polysaccharide (Persson et al. above) in a two step process. Thus selectivity over target and contaminant protein (as well as second polymer) occurs in the first partition step, followed by use of temperature induced phase separation (of typically EOPO polymer rich phase) to isolate target and polymer into target containing aqueous phase floating on top of a self-associated polymer rich denser phase. In regard to the use of thermoseparating phases on their own (i.e. one polymer but lower salt concentration) systems the general wisdom has been that they tend to not be useful as they offer little selectivity and should be used in systems with other polymers. A distinguished international research group was led to conclude “the water EOPO system is therefore only suitable for partitioning of hydrophobic molecules (such as denatured proteins or tryptophan-rich peptides) or for solution concentration by selective water removal (similar arguments hold for the micellar two-phase systems)” (Hans-Olof Johansson, Gunnar Karlström, Folke Tjerneld, Charles A. Haynes, J. Chromatography B, 711 (1998) 3-17). In regard to such applications the effect of various salts and other additives on phase separation of another EOPO polymer (Breox 50 A 1000 a random copolymer consisting of 50% ethylene oxide and 50% propylene oxide, molecular mass number average 3900, Specialty Chemicals, Southhampton, UK). were studied by Cunha et al. (Maria Teresa Cunha, Folke Tjerneld, Joaquim M. S. Cabral, Maria Raquel Aires-Barros, Journal of Chromatography B, 711 (1998) 53-60).
Teixeira et al. (Martinha Pereira, You-Ting Wu, Armando Venancio, José Teixeira, Biochemical Engineering Journal 15 (2003) 131-138) investigated the partitioning of endo-polygalacturonase (endo-PG) in systems composed of UCON 50-HB in two-polymer systems together with polyvinylalcohol, or hydroxypropylstarch or with relatively high concentrations of ammonium sulfate. The latter system required heating to effect formation of two phases but was the most promising in terms of reagent cost and ability for reagents to be added to culture (again clarified) supernatant prior so that 70% of the final system consisted of clarified culture broth. The UCON polymer could be recycled in a three step process in which endo-PEG was concentrated ten times and 95% of enzyme activity was recovered. In regard to this work two observations are note worthy. First the 5% minimum ammonium salt concentration (50 g/L or approx. 0.38M) necessary to effect formation of two phases is still significant and required 10% UCON. Raising temperature to 40° C. only decreased these values to 3% (0.23 M) salt and 5% polymer. So the system still contained significant added salt. Secondly at temperatures above 30 degrees Texeira et al noted phase inversion in their systems so that the top, polymer poor, less-dense phase at room temperature became the bottom phase. Such effects while interesting, could pose problems in regard to large scale processing particularly in systems containing cells and cell debris which would tend to sediment. In addition to the above noted thermoseparating phase systems there are a wide range of thermoseparated micellular systems involving hydrophobically modified EOPO and similar polymers (for discussion see H.-O. Johansson et al, 1998 above). Many patents related to the above two polymer thermoseparating aqueous phase systems are currently held by G.E. Healthcare, a General Electric company.
The ability of PEG and salt two-phase systems to partition cells and cell debris to interface, and therefore for possible use of phase partitioning to effect partial clarification, has been known for some time. Köhler et al. formed 7.5% w/w PEG 1500 and 14% potassium phosphate two-phase systems directly in a bioreactor and used them to purify a recombinant protein in E. coli (Kristina Köhler, Björn Nilsson, Andres Veide, Recovery of extracellular human insulin-like growth factor-I and II as a fusion protein from Escherichia coli culture broth by aqueous two-phase extraction, Bioseparation, 3 (1992-1993) 241-250) noting approximately 90% of cells were not in the target containing phase. However batch centrifugation was still used in the process to effect complete phase separation and was advocated, in continuous centrifugation mode, for larger scale applications. Such polymer-high salt systems have much greater interfacial tension than polymer-polymer systems formed at lower salt concentration and may be expected to function to effect some clarification due to their relatively high interfacial tension. However they would supposedly still be limited in terms of capacity (target solubility) due to the high salt concentrations required. Köhler et al. noted that biomass added to system affected some partition results. Since most studies related to finding optimized systems for recombinant protein (esp. antibodies) processing have been done using clarified feed the systems found may not be optimal or even function for clarified feed. That is why several examples of unclarified feed were used in the present work.
It can be seen from the above discussion that two phase partitioning holds much promise as method for primary processing (clarification and target partial purification) of various substances such as proteins including biopharmaceuticals from complex feeds streams however to date certain challenges have not been overcome. These include cost of reagents (polymer and salts, or two polymers), capacity issues related to needed dilution of target containing process streams, possible need to add various affinity substances (e.g. TEG-COOH) to increase target partition, removal of phase system forming substances prior to or during further downstream processing steps, and modifying target containing phases to allow for further downstream processing. From the point of simplicity thermoresponsive polymer and water systems (which do not involve micelle formation or use of special hydrophobically modified thermoresponsive polymers) may be the most attractive as they are typically neutral and in some cases biocompatible. So too residual polyethoxy and other polymers in target containing phase may not only be seen as relatively inert substances. They may confer some advantages for a. further processing by multistep partitioning, b. spray drying of target containing solution (e.g. Jessica Elversson, Anna Millqvist-Fureby, Aqueous two-phase systems as a formulation concept for spray-dried protein, International Journal of Pharmaceutics 294 (2005) 73-87) and, due to their well known antifreeze and antioxidant properties, c. low temperature intermediate storage of target containing phase solution prior to further processing. However established wisdom and experience has been that their formation required relatively high polymer and salt concentrations and the phases formed offered little selectivity, required fairly high salt concentrations and might not work to effect clarification.
For many years biopharmaceutical fermentation, purification and polishing/formulation have been seen as separate process areas. A major reason for this was they often involved different unit operations and volume scales. Both of these were related to the concentration of target substance and inversely the process volumes handled in different processing stages. Thus fermentation at perhaps 1 mg/mL, purification by affinity or ion exchange raising the concentration to perhaps 30 mg/mL with polishing followed by formulation steps taking the target to 100 in liquid (mg/mL) or solid (mg/g) form. As a result initial processing steps might involve process volumes 100× larger than formulation steps. These distinctions are blurring now that antibodies and other biopharmaceuticals can reach 30 mg/mL in fermentation feed and early ion exchange or other purification steps achieve 100 mg/L. Formulation often involves combining protein or other biopharmaceutical with excipients such as polymers including Dextrans™, poly(ethylene glycol)s or Polysorbates™ (polyethoxylated sorbitan and laurate) and various commercially available copolymers or block copolymers of oxyethylene or oxypropylene such as Tergitols™ or Pluronics™. Excipients can be charged including use of other proteins (i.e. charged amphipathic biopolymers) such as albumin. Excipients stabilize the biopharmaceutical during storage, maintain high concentrations without inducing aggregation, and allowing for rapid dissolving and uptake in the body. Some polymeric or other excipients may also enhance not only the delivery but the pharmacological properties of drugs via for example adjuvant action. Given the above it is natural that any partition, precipitation or other unit operation method which localizes antibodies or other target proteins in solution, or insoluble complex, with biocompatible polymers should be of interest not only in regard to purification but also formulation, storage, delivery and efficacy of biopharmaceuticals. Especially as polymers such as those noted above are often found in antibody and other pharmacological formulations. One key point is that any commercially viable method must be able to handle complex feeds which include proteins or other targets at relatively high concentrations (e.g. >10 g/L), and process them without significant (i.e. >2×) dilution. The above considerations hold not only for recombinant protein, nucleic acid and other biopharmaceuticals but also for vaccines, and other biotherapeutics and bioparticles.
Vaccines, and especially viral vaccines pose a set of interesting processing challenges illustrated by the processing of influenza vaccines. Much flu vaccine is produced in eggs. This offers the interesting challenge of removing ovalbumin protein and other contaminants from the viral targets. This is often done via sucrose density gradient centrifugation. However modern processing is going more and more over to processing of viral vaccines in cells (typically MDCK or Vero kidney cell lines) grown either in suspension culture or adherent culture where the cells grow attached to colloidal carriers. In both cases the cultured cells are infected with virus, which propagate to the point where the cells either lyse naturally or are readily lysed by various chemical or physical treatment. In both cases the end results is a complex feed which contains various larger (>1 micron) particles, cell debris, intact virus (which is the target to be purified) and virus related debris such as cell membrane fragments containing viral proteins. Following use of centrifugal or other methods to remove cells and related debris, sucrose density gradients may be employed to separate the viral related fractions into intact and debris fractions. Such methods are of course decades old technology and there have been attempts to employ newer separation methods such as aqueous polymer two phase partitioning or column chromatography. Most work involving partitioning of viruses was done over ten years ago and has been reviewed by Lena Hammar (Lena Hammar, Concentration of Biomaterials: Virus Concentration and Viral Protein Isolation, Chapter 62, pp. 627-658, in Methods in Enzymology, Volume 228, Aqueous Two-Phase Systems, H. Walter and G. Johansson, Eds., Academic Press, New York, 1994) where she noted that “Extraction in aqueous polymer systems remains an attractive option when virus purification from large volumes is involved and in dealing with labile viruses”. Hammar and related references provide many examples of partitioning of a wide variety of different viruses of medical significance. The labile nature of viruses generally dictate that two polymer (typically PEG and dextran) phase systems, which offer lower interfacial tensions than single polymer and high salt (e.g. PEG and sodium phosphate) systems were used. Naturally fractionation of virus using such systems suffers many of the same drawbacks which are related to processing antibodies or other macromolecular targets via partitioning. This includes cost of two polymers, and addition of a separate partitioning step to a process. Some vaccine processes recovery of viral product after centrifugal clarification followed by sucrose density gradient fractionation can be as low as 20%. An inexpensive partition system which offered as good or better recovery while replacing one or both of the clarification and density gradient steps is desirable, especially if it could be performed in disposable bag format, rather than in fixed line centrifuges. Commercial viability of partition processing of viral vaccines must also rest on new inexpensive systems which offer excellent selectivity. Such a goal is achieved in this application.